The impact of loss of function DNA sequence variants in the human protocadherin gene cluster on neural circuit assembly.

人类原钙粘蛋白基因簇中功能丧失 DNA 序列变异对神经回路组装的影响。

• 批准号: 10736632
• 负责人: THOMAS P MANIATIS
• 金额: $ 74.86万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736632
• 关键词: Affect; Alleles; B-Lymphocytes; Bar Codes; Behavior; Binding; Binding Proteins; Biological; Biological Assay; Bipolar Disorder; Brain; CRISPR/Cas technology; Cell Aggregation; Cell Communication; Cell Culture Techniques; Cell Line; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Cellular Assay; Chromatin; Collaborations; Complex; DNA Sequence; DNA sequencing; Data; Defect; Development; Disease; Dominant-Negative Mutation; Dorsal; Epilepsy; Etiology; Failure; Family; Future; Gene Cluster; Gene Expression; Genes; Genetic; Genetic Risk; Goals; Guide RNA; Human; Human Genetics; In Vitro; Individual; Intellectual functioning disability; Joints; K562 Cells; Knockout Mice; Knowledge; Link; Major Depressive Disorder; Mammals; Membrane; Methods; Microcephaly; Modeling; Molecular; Mus; Mutation; Nature; Neurites; Neurodevelopmental Disorder; Neurons; Pathogenesis; Pathway interactions; Pattern; Play; Privatization; Protein Isoforms; Proteins; Regulation; Role; Schizophrenia; Single Nucleotide Polymorphism; Structure; Surface; Synapses; Testing; Therapeutic; Therapeutic Intervention; Transfection; Variant; Work; anxiety-related behavior; autism spectrum disorder; causal variant; cell transformation; cohort; excitatory neuron; functional restoration; gain of function; gene function; genetic association; genome-wide; in vivo; induced pluripotent stem cell; inhibitory neuron; insight; knock-down; loss of function; lymphoblastoid cell line; mutant; neural; neural circuit; neural network; neuronal circuitry; neuropsychiatric disorder; novel; novel therapeutic intervention; prevent; proband; protein structure; targeted treatment; trafficking; transforming virus; variant detection

REVISED PROJECT SUMMARY/ABSTRACT: Complex neural circuits are accurately assembled between the approximately 80 billion neurons in the human brain during development. The mammalian clustered protocadherin (Pcdh) genes, which encode a family of highly diverse cell-surface homophilic binding proteins, provide individual neurons with a unique cell-surface identity, or barcode required for normal neural circuit assembly. Previous structural, functional, and gene expression studies of the clustered Pcdh genes and proteins have revealed complex mechanisms by which Pcdh barcodes are generated and function, as well as the impact of loss of function Pcdh mutations on neural circuit assembly and behavior in mice. A remarkable feature of the Pcdh barcode is that it consists of a cell surface protein “lattice” on the membranes of interacting neurons. The Pcdh barcode has been shown to play a critical role in neurite self-avoidance, neuronal tiling and normal behavior in mice. Dominant loss of function Pcdh mutations that disrupt cell-cell interactions have been demonstrated in cell culture studies. Although the failure to accurately assemble neural circuits has been linked to neurodevelopmental and neuropsychiatric disorders, the occurrence of single nucleotide variants (SNVs) in individual PCDH genes across the PCDH gene cluster do not meet a genome wide statistical association criteria for genetic association in large scale DNA sequencing studies of autism spectrum disorder (ASD) cohorts. However, advances in understanding the regulation and function of the PCDH gene cluster and the recent availability of lymphoblastoid cell lines (LCLs) from ASD probands in the Simons Simplex cohort bearing disruptive de novo SNVs in PCDH genes presents a unique opportunity to further explore PCDH gene function in human neurons derived from human induced pluripotent stem cells. The aims are to conduct a systematic analysis of de novo SNVs across the PCDH gene clusters identified in ASD cases. In Aim 1 we will determine how SNVs in PCDH isoforms impact homophilic interactions and intracellular localization of PCDH proteins in cell-based in-vitro homophilic binding assays. In Aim 2, we will generate CRISPR-Cas9 edited hiPSCs bearing loss of function PCDH mutations and differentiate them into neurons to assess deficits in self-avoidance in the hiPSC-derived neurons. In Aim 3, we will investigate the impact of SNVs in PCDH isoforms on neural circuits and functional connectivity in mice, using serotonergic neurons as a model. These proposed studies should significantly advance our understanding of the functional role of the PCDH locus in neural circuit assembly during brain development.

修订后的项目摘要/摘要： 在发育过程中，人脑中约 800 亿个神经元之间精确地组装了复杂的神经回路。哺乳动物簇状原钙粘蛋白 (Pcdh) 基因编码高度多样化的细胞表面同亲结合蛋白家族，为单个神经元提供独特的细胞表面身份或正常神经回路组装所需的条形码。先前对聚集的 Pcdh 基因和蛋白质的结构、功能和基因表达研究揭示了 Pcdh 条形码生成和功能的复杂机制，以及功能丧失的 Pcdh 突变对小鼠神经回路组装和行为的影响。 Pcdh 条形码的一个显着特征是它由相互作用的神经元膜上的细胞表面蛋白“晶格”组成。 Pcdh 条形码已被证明在小鼠神经突自我回避、神经元平铺和正常行为中发挥着关键作用。细胞培养研究已证明显性功能丧失 Pcdh 突变会破坏细胞间相互作用。 尽管无法准确组装神经回路与神经发育和神经精神疾病有关，但在自闭症谱系障碍 (ASD) 群体的大规模 DNA 测序研究中，PCDH 基因簇中各个 PCDH 基因中单核苷酸变异 (SNV) 的发生不符合基因组范围内遗传关联的统计关联标准。然而，在了解 PCDH 基因簇的调控和功能方面取得的进展，以及最近从 Simons Simplex 队列中自闭症谱系障碍 (ASD) 先证者中获得 PCDH 基因中具有破坏性从头 SNV 的淋巴母细胞系 (LCL)，为进一步探索源自人类诱导多能干细胞的人类神经元中的 PCDH 基因功能提供了独特的机会。 目的是对自闭症谱系障碍 (ASD) 病例中发现的 PCDH 基因簇中的从头 SNV 进行系统分析。在目标 1 中，我们将确定 PCDH 亚型中的 SNV 如何影响基于细胞的体外同质结合测定中 PCDH 蛋白的同质相互作用和细胞内定位。在目标 2 中，我们将生成 CRISPR-Cas9 编辑的具有 PCDH 功能丧失突变的 hiPSC，并将其分化为神经元，以评估 hiPSC 衍生神经元的自我回避缺陷。在目标 3 中，我们将使用血清素能神经元作为模型，研究 PCDH 亚型中的 SNV 对小鼠神经回路和功能连接的影响。这些拟议的研究将显着促进我们对 PCDH 基因座在大脑发育过程中神经回路组装中的功能作用的理解。